A basic peroxidase isoenzyme from vacuoles and cell walls of Vitis vinifera

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A basic peroxidase isoenzyme from vacuoles and cell walls of Vitis vinifera
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  Pergamon PII: S0031-9422 96)00825-4 Phytochemistry, Vol. 45, No. 2, pp. 22%232, 1997 © 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0031-9422/97 17.00+0.00 A BASIC PEROXIDASE ISOENZYME FROM VACUOLES AND CELL WALLS OF VITIS VINIFER M. MORALES and A. Ros BARCEL6* Department of Plant Biology, University of Murcia, E-30100 Murcia, Spain Received 16 Auyust 1996) Key Word Index--Vitis vinifera; vitaceae; basic peroxidase isoenzyme; cell wall; vacuole; kinetic properties; phenolic oxidation. Abstract--The substrate profile of a basic peroxidase isoenzyme located in vacuoles and cell walls from Vitis vinifera and efficacy in oxidizing both vacuolar and cell wall phenolic substrates was studied. The reactivity of this isoenzyme with H202 [k~ (Col formation constant) = 1.1 (quercetin), 1.6 (myricetin), 1.7 trans-resveratrol) and 10.4 (coniferyl alcohol) #M -l sec-1] and with the phenolics [k3 (Coil reduction constant) = 20.7 (quercetin), 6.9 (myricetin), 11.9 trans-resveratrol) and 8.7 (coniferyl alcohol) #M -I sec-q, suggests that the isoenzyme reacts with H202 with a similar reactivity to that shown by other peroxidases, and that both vacuolar and cell wall phenolics are excellent substrates for Coil reduction. © 1997 Elsevier Science Ltd. All rights reserved INTRODUCTION Peroxidase (EC 1.11.1.7) is the enzyme responsible for the cross-linking of phenolic moieties during the biosynthesis of lignins in the plant cell wall [1-3]. As may be expected from its specific role in lignin biosynthesis, peroxidase is mainly located in the cell wall, although other subcellular localizations have been reported: for example, in the vacuole. When we have studied peroxidase isoenzyme localization in Lupinus albus (Fabaceae) [4, 5], Vitis vinifera (Vit- aceae) [6, 7], Capsicum annuum (Solanaceae) [8]; Lac- tuca sativa (Asteraceae) [9], and Catharanthus roseus (Apocynaceae) [10], we found that, in all the studied plant materials, the cell wall contains the complete set of peroxidase isoenzymes present in the plant cell, including both basic (pI > 7.0) and acidic (pI > 7.0) peroxidase isoenzymes; while vacuoles only contain the basic peroxidase isoenzyme group of high pI (pI > 9.2) (BPrx HpI), an isoenzyme group also detected in the cell wall. These results are not surpris- ing, since a similar dual localization for the most basic peroxidase isoenzyme has been found in other plant species [11]. Although some metabolic analogies have been found in plant cells between vacuoles and cell walls, especially regarding both subcellular compartments as lytic compartments [12], the fact that the BPrx HpI isoenzyme may be located in vacuoles and cell walls raises a question on its metabolic plasticity, since the *Author to whom correspondence should be addressed. available substrates in these two subcellular com- partments are somewhat different. In the case of grapevines (Vitis vinifera), the BPrx HpI isoenzyme is located at vacuole level in hypo- dermal cells and at cell wall level in the xylem [7, 13, 14]. In this first case, this isoenzyme appears to play a role in the metabolic turnover of certain flavonol glycosides (quercitrin and myricitrin) once the agly- cones (quercetin and myricetin) have been released by specific glycosidases [15]; while at the xylem cell wall level it is more probable that it plays a role in the polymerization of cinnamyl alcohol to lignins [16]. For this reason, in this work we have studied the catalytic efficiency of this isoenzyme during the oxi- dation of the aglycones (i.e. quercetin and myricetin) of vacuolar-type substrates (quercitrin and myricitrin) and of cell wall-type substrates (i.e. coniferyl alcohol). Furthermore, the catalytic efficiency of this isoenzyme for these substrates is compared with its catalytic efficiency during the oxidation of trans-resveratrol (a particular phenolic located in both vacuoles and cell walls). From a comparison of the values obtained for the constants of compound I formation (k0 and compound II reduction (k3), a strong metabolic plas- ticity for this BPrx HpI isoenzyme is suggested, which is in accordance with their two different subcellular locations. RESULTS Oxidation of the phenolics, quercetin, myricetin, trans-resveratrol and coniferyl alcohol, by the grape- 229  230 M. MORALES nd A. Ros BARCEL6 oo A o.o~ /- 0 00 0 0 0.2 0.4 0.6 B Fig. I. Plot of parameters A (nmol s -~) vs B (mM) obtained by varying H202 for three quercetin concentrations [0.01 mM (1), 0.05 mM (2) and 0.15 mM (3)]. From the slope of the straight line (2 [E] k0, k~ was calculated to be 1.1 #M -' s -~. vine BPrx HpI isoenzyme follows Michaelis-Menten type kinetics, with inhibition by substrate at higher concentrations. To calculate the microscopic con- stants (k~ and k3) for the oxidation of phenolic sub- strates by this basic peroxidase isoenzyme fraction, the rates of oxidation of phenolics were fitted for each concentration of phenolic and H202, according to the generally accepted mechanism for the peroxidase reac- tion [17]: kl P+H20 2 ...... CoI+H20 (1) k2 Col + RH ........ Coil + R (2) k3 Coil + RH , P + R (3) where RH is the particular phenolic and Col and Coil are the key intermediates in the peroxidase cycle, compound I and compound II. For non-inhibitory concentrations, the initial rates of the phenolic oxidation may be fitted to the fol- lowing equation: A[H202] V-- B+ [H202] where `4 = 2 [E] k3 [RH] and B = k3/k,) [RH]. Dou- ble-reciprocal plots 1/v vs 1/[H202]) allow us to cal- culate A and B values for each phenolic concentration. Fig. 1 shows the plot of A vs B values for three quer- cetin concentrations. From this straight line (A = 2 [El kjB), it is possible to calculate the k~ value (Com- pound I formation constant) for the peroxidase- mediated quercetin oxidation. The value obtained, as well as those obtained for the peroxidase-mediated myricetin, trans-resveratrol and coniferyl alcohol oxi- dation, are shown in Table 1. Similarly, the dependence of v on [RH] may be written as: Table 1. k, (#M-' sec -;) and k3 (#M -; sec-') values at 25 ° for the oxidation of several phenolic substrates by the basic peroxidase isoenzyme from Vitis vinifera Substrate pHopt k~ k3 Quercetin 4.0 I. 1 20.7 Myricetin 6.0 1.6 6.9 trans-Resveratrol 4.0 1.7 11.9 Coniferyl alcohol 5.0 10.4 8.7 A[RH] t) B+[RH] where A = 2 [E] k, [H202] and B = kdk3) [H202]. Double reciprocal plots (1/v vs 1/[RH]) allow us to calculate A and B values for each H202 concentration. Figure 2 shows the plot of ,4 vs B values for three H202 concentrations during the oxidation of myrice- tin. From this straight line (.4- 2 [El k3 B), it is possible to calculate the k3 value (Compound II reduction constant) for the peroxidase-mediated myr- icetin oxidation. The value obtained, as well as those obtained for quercetin, trans-resveratrol and coniferyl alcohol, are shown in Table 1. In all the cases, k, and k3 values were calculated at the optimum pH for the peroxidase-catalysed oxidations, these values also being listed in Table 1. From these results, it may be concluded that oxi- dation of these phenolics by the grapevine BPrx HpI isoenzyme follows the accepted model for peroxidase oxidation, in which CoI and CoII appear as the main intermediates in the catalytic cycle [eqns (1)-(3)]. This observation is supported by the good fits to a straight line which passed through the coordinate origin when the parameters ,4 vs B were represented for varying phenolics (Fig. 1) and H/O2 (Fig. 2) concentrations. A 0.3 0.2 0.1 i/e= / 0.0 0.0 @ I 0.1 0.2 B Fig. 2. Plot of parameters A (nmol s -I) vs B (mM) obtained by varying myricetin for three H202 concentrations [0.25 mM (1), 0.50 mM (2) and 2.0 mM (3)]. From the slope of the straight line (2 [E] k3), k3 was calculated to be 6.9/~M-' S-'.  Substrate profile of a basic peroxidase 231 DISCUSSION From the rate constant values (k~) shown in Table 1, it can be deduced that the grapevine BPrx HpI isoenzyme is capable of oxidizing vacuolar phenolic substrates (i.e. quercetin, myricetin, and trans-resver- atrol) as well as cell wall phenolic substrates (i.e. trans- resveratrol and coniferyl alcohol) without strong sub- strate discrimination. Examination of the Col formation constant (k0 for the four substrates assayed reveals that it varies from 1.1 to 1.7/~M -~ sec -t for polyhydroxy phenolic com- pounds (i.e. quercetin, myricetin and trans-resver- atrol), while it reaches the value of 10.4/~M -~ sec -~ for a guaiacyl-type phenolic compound (i.e. coniferyl alcohol). Although, a priori, it is difficult to explain at molecular level how the nature of the substrate influences the rate constant of Col formation, this is a fact described in the literature [17]. Moreover, this value is usually greater for guaiacyl-type structures than for polyhydroxy phenolic compounds [17]. As has been discussed above, kl values depend on the nature of the reducing substrate. For this reason, it is misleading to compare the k~ values obtained in this work using the grapevine BPrx HpI isoenzyme (see Table 1) with those obtained with different sub- strates for other peroxidase preparations. A previous study using a moderately basic peroxidase isoenzyme from barley [17] at pH 3.96 showed a value for k~ of 6.7/~M -~ sec -~ using coniferyl alcohol as substrate. If we compare this value with that obtained for the grapevine BPrx HpI isoenzyme (Table 1), one may conclude that the reactivity of the grapevine BPrx HpI isoenzyme with H202 [whose magnitude is evaluated through the constant of Col formation (k0] is greater than that shown by barley peroxidase during the oxi- dation of coniferyl alcohol oxidation. In the case of the cell wall-located phenolic compound, coniferyl alcohol, in which the k3 value may be compared with that shown in literature, the reactivity of the grapevine BPrx HpI isoenzyme for coniferyl alcohol is higher than that shown by other peroxidases. Thus, k3 values reported for coniferyl alcohol are 2.80/~M-1 sec-~ for the moderately basic peroxidase isoenzyme from horseradish at pH 4.5 [ 18], and 2.4 gM -~ sec -t for the moderately basic per- oxidase isoenzyme from barley at pH 3.96 [17], values clearly below that shown by the grapevine BPrx HpI isoenzyme for coniferyl alcohol (8.3 #M -~ sec -1, Table 1). On the other hand, coniferyl alcohol is frequently recognized as one of the best substrates for Coil reduction [17, 18]. However, the grapevine BPrx HpI isoenzyme oxidizes some vacuolar phenolic substrates (i.e. quercetin, myricetin and trans-resveratrol) with rate constants comparable, and even greater, than that shown for coniferyl alcohol (Table 1). These results illustrate the great reactivity and metabolic plasticity for oxidizing phenolic substrates of an isoenzyme found in two different subcellular localizations (vacu- ole and cell wall), where the availability of particular substrates, conditioned by its tissue-specific local- ization, appears to be the main factor delimiting its metabolic function. EXPERIMENTAL Chemicals. Quercetin (3,3',4',5,7-pentahy- droxyflavone) and myricetin (3,Y,4',5,5',7-hexa- hydroxyflavone) were purchased from Extrasynthese (Genay, France). Coniferyl alcohol (3-methoxy, 4- hydroxycinnamic) and H202 from Sigma Trans- resveratrol [(E)-3',4,5'-trihydroxystilbene] was syn- thesized by a Wittig reaction linking 3,5-dime- thoxybenzyltriphenyl phosphine bromide and p-ani- saldehide [19]. Methylated precursors were used to protect the OH groups, which were removed by boron tribromide. The Wittig products were a mixture of (E) and (Z) isomers. The E)/ Z) mixture was isomerized to the pure (E) isomer by heating with a catalytic amount of diphenyl disulfide in tetrahydrofuran. The identities of the structures were confirmed from their 1H-NMR spectra: the coupling constants of the ole- finic protons were 16-17 Hz for the (E)-isomer and 12 Hz for the (Z)-isomer. Confirmation of trans con- formation was deduced from their UV-spectral characteristics (~taO = 307 rim), IR absorption peak at 965 (w) cm-1 trans form of the double bond), 6 [tH-NMR (CDC13, CD3COCD3, 300 MHz)] = 6.79- 6.84 (m, 3H, H3, +Ha/Hb) and 6.97 (d, IH, J = 16.5 Hz, Ha/Hb) and ~ [t3C-NMR (CDC13, CD3COCD 3, 75 MHz)] = 125.07 (CJCb) and 127.38 (Ca/Cb). All the other chemicals used in this work were obtained from various commercial suppliers and were of the highest purity available. Plant material and peroxidase isoenzyme puri- fication. The Vitis vinifera cultivar, Gamay rouge, was grown in field and sampled in August at veraison. Fruit clusters were transported to the laboratory and frozen immediately at -30 °. From this material, the homogenization and purification of the BPrx HpI iso- enzyme by prep. isoelectric focusing have been described in refs [15, 16]. Examination by analytical isoelectric focusing of the purified isoenzyme revealed that it migrates as a sole peroxidase isoenzyme band during electrophoresis [16]. Spectrophotometric studies. The spectrophoto- metric assays for the oxidation of phenolics by the grapevine BPrx HpI isoenzyme were performed at 25 ° in a reaction medium containing variable concs of phenolics [0.01, 0.05 and 0.15 mM for quercetin; 0.025, 0.05 and 0.1 mM for myricetin and coniferyl alcohol; and 4.5, 45 and 90/~M for trans-resveratrol] and H202 [0.1-5.0 mM] in 0.1 M Tris-acetate buffer. The reaction was initiated by the addition of grapevine peroxidase. Oxidation of quercetin, myricetin, trans- resveratrol and coniferyl alcohol were monitored by the changes in absorbance using a A~365 = 13200, Ag365 ---~ 7200, A~306 = 26800 and Ag260 -2200 M -l cm- 1, respectively. The concn of peroxidase was deter-  232 M. MORALES nd A. Ros BARCeL6 mined at 403 nm using a e4o3 = 1.02 105 M -~ cm -~ for the Soret band. To calculate the microscopic constants (kl and k3) for the oxidation of phenolic substrates by this basic peroxidase isoenzyme fraction, the rates of oxidation of phenolics were fitted for each concn of phenolic and H202, according to the generally accepted mech- anism for the peroxidase reaction [17]: k~ P+H202 ~ CoI+H20 (l) Col+ RH k2 , CoII + R 2) k3 Coil + RH , P + R (3) where RH is the particular phenolic and Col and Coil are the key intermediates in the peroxidase cycle, compound I and compound II. Assuming that k2 > k3, as occurs for most peroxidases [17], the steady-state equation rate may be written as: 2[E]k3[RH][H202] v= (4) k3/kl)[Rn] + [H202] from which the dependence of v on [H202] may be written as: A[H202] v - (5) B + [H202] where A = 2 [El k3 [RH] and B = k3/kl) [RH]. Dou- ble-reciprocal plots 1/v vs 1/[H202]) allow us to cal- culate A and B values for each phenolic concn. Plot- ting A vs B values gives a straight line (A = 2 [El kl B), and from its slope it is possible to calculate the kl value. Similarly, and starting from eqn (4), the dependence of v on [RH] may be written as: A[RH] v - -- (6) B+[RH] where A = 2 [E] kl [H202] and B = k3/k3) [H202]. Double reciprocal plots 1/v vs 1/[RH]) allow us to calculate A and B values for each H202 concn. Plotting A vs B values gives a straight line (A = 2 [E] k3 B), and from its slope it is possible to calculate the k3 value. These A/B plots allows us to calc. true reaction constants (ki) from steady-state measurement of the oxidation rate avoiding their dependence on substrate concn. [17]. obs From eqn. 4, the catalytic efficacy keat/KM) for the utilization of H202 would be given by: k=,t/K~S = 2k3[RH] _ 2k, k3/k,)[RH] while the catalytic efficacy k~,t/K~ s) for the utilization of the phenolic would be given by: k~t/K~S 2k,[H202] kl/k3)[H202 ] = 2k3 Acknowledgements--This work has been partially supported by grants from the CICYT (Spain), Projects no. ALl 93/573 and ALI 95/1018. M. Morales holds a fellowship from the MEC (Spain). REFERENCES 1. Ferrer, M. A., Pedrefio, M. A., Mufioz, R. and Ros Barcel6, A., FEBS Letters, 1990, 276, 127. 2. Lagrimini, L. M., Vaughn, J., Erb, A. and Miller, S. A., HortSeience, 1993, 28, 218. 3. Ros Barcel6, A., Protoplasma, 1995, 186, 41. 4. Ros Barcel6, A., Ferrer, M. A., Garcia-Flor- enciano, E. and Mufioz, R., Botanica Acta, 1991, 104, 272. 5. Ferrer, M. A., Pedrefio, M. A., Ros Barcel6, A, and Mufioz, R., Journal of Plant Physiology, 1992, 139, 611. 6. Garcia-Florenciano, E., Calder6n, A. A., Pedrefio, M. A., Mufioz, R. and Ros Barcel6, A., Plant Growth Regulation, 1991, 10, 125. 7. Calder6n, A. A., Garcia-Florenciano, E., Pedrefio, M. A., Mufioz, R. and Ros Barcel6, A., Zeitschrift far Naturforschung, 1992, 47e, 215. 8. Bernal, M. A., Pedrefio, M. A., Calder6n, A. A., Mufioz, R., Ros Barcel6, A. and Merino de Chc- eres, F., Annals of Botany, 1993, 72, 415. 9. G6mez-Tena, M., Pedrefio, M. A., Ros Barcel6, A. and Ferrer, M. A., Journal of the American Society for Horticultural Science, 1994, 119, 1276. 10. Sottomayor, M., de Pinto, M. C., Salema, R., DiCosmo, F., Pedrefio, M. A. and Ros Barcel6, A., Plant Cell and Environment, 1996, 19, 761. 11. Schloss, P., Walter, C. and Mader, M., Planta, 1987, 170, 225. 12. Wink, M., Journal of Experimental Botany, 1993, 44, 231. 13. Calder6n, A. A., Zapata, J. M., Mufloz, R. and Ros Barcel6, A., HortScience, 1993, 28, 38. 14. Calder6n, A. A., Zapata, J. M. and Ros Barcel6, A., Plant Cell Tissue and Organ Culture, 1994, 37, 121. 15. Morales, M., Pedrefio, M. A., Mufioz, R., Ros Barcel6, A. and Calder6n, A. A., Journal of Sci- ence and Food Agriculture, 1993, 62, 385. 16. Morales, M., Pedreflo, M. A., Mufioz, R., Ros Barcel6, A. and Calder6n, A. A., Food Chemistry, 1993, 48, 391. 17. Rasmussen, C. B., Dunford, H. B. and Welinder, K. G., Biochemistry, 1995, 34, 4022. 18. Marklund, S., Ohlsson, P. I., Opara, A. and Paul, K. G., Biochimica et Biophysica Acta, 1974, 350, 304. 19. Morales, M., Alcfintara, J. and Ros Barcel6, A., American Journal of Enology and Viticulture, 1997, in press.
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